A split protease-E. coli ClpXP system quantifies protein-protein interactions in Escherichia coli cells

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Characterizing protein-protein interactions (PPIs) is crucial for understanding protein function.
• Existing methods for PPI analysis have limitations in scope or application.
• CRISPR/Cas systems are essential for microbial immunity and gene editing technologies.

Purpose of the Study:

• To develop a novel, efficient, and broadly applicable single-cell methodology for exploring protein-protein interactions (PPIs).
• To characterize the interactions within type I-E and I-F CRISPR/Cas effector complexes.
• To enhance the understanding of CRISPR/Cas complex functions and structures.

Main Methods:

• Development of the split protease-E. coli ClpXP (SPEC) method, integrating a split human Rhinovirus 3C (HRV 3C) protease, super-folder GFP (sfGFP), and ClpXP-SsrA protein degradation machinery.
• Utilizing a highly efficient and re-assemblable split HRV 3C protease within the SPEC framework.
• Converting cellular PPIs into quantitative fluorescence signals via a protease-mediated proteolytic reaction.

Main Results:

• The SPEC method demonstrated high efficiency and broad temperature adaptability for converting PPIs into fluorescence signals.
• Successfully identified and characterized interactions among effectors of type I-E and I-F CRISPR/Cas complexes.
• The method provides a robust platform for studying PPIs in both eukaryotic and prokaryotic species within E. coli cells.

Conclusions:

• The SPEC methodology offers a powerful new tool for high-throughput screening and analysis of PPIs in single cells.
• The study provides novel insights into the functional mechanisms and structural organization of CRISPR/Cas effector complexes.
• SPEC facilitates a deeper understanding of protein interactions, contributing to advancements in molecular biology and biotechnology.